The present invention relates to an enzyme electrode unit, and more particularly to an enzyme electrode unit in which diffusion-limiting membranes limit the diffusion of a target substance to be measured and the substance as limited in diffusion is guided to an enzyme-immobilized membrane.
It is known that a physiologic active substance has a characteristic capable of selectively detecting a very complicated organic compound, protein or the like with high sensitivity. With attention directed to this characteristic, researches and developments have been made on measurement of such organic compound, protein or the like with the use of an enzyme electrode unit having base electrodes on which a physiologic active substance is immobilized.
When measuring a target substance with the use of the enzyme electrode unit above-mentioned, the target substance is oxidized or reduced by the enzyme immobilized on the surfaces of the base electrodes. By measuring the concentration of the oxygen, hydrogen peroxide or the like which undergoes a change by such oxidization or reduction, the concentration of the target substance can be indirectly measured.
For example, when the concentration of glucose is to be measured, glucose oxidase (hereinafter referred to as GOD) may be used as a physiologic active substance. In this case, the following reaction takes place: ##STR1## Accordingly, the concentration of glucose can be determined by detecting the decrease in oxygen concentration or the increase in hydrogen peroxide concentration.
More specifically, an enzyme-immobilized membrane having enzyme immobilized on an acetylcellulose membrane is stuck to the surfaces of hydrogen peroxide electrodes used as the base electrodes, and a polycarbonate membrane covers the enzyme-immobilized membrane. To enhance the sensitivity of measuring the concentration of glucose, the total thickness of both membranes is set to 10 .mu.m.
In the example above-mentioned, the total membrane thickness is extremely thin in order to enhance the concentration measuring sensitivity. Consequently, the solution is guided to the enzyme-immobilized membrane with the glucose concentration being extremely high. Further, as apparent from the reaction formula mentioned earlier, the glucose concentration measuring limit is determined according to the amount of oxygen contained in a target solution to be measured. As a result, the glucose concentration measuring limit is very low. In this connection, if it is intended to increase the glucose concentration measuring limit, it is required to previously dilute the glucose solution at a predetermined dilution ratio. This causes the dilution mechanism to be complicated, and requires an expensive dilution apparatus.
To overcome such problems, it has been proposed, as disclosed in the Japanese Laid-Open Patent Publication No. 59-22620, that a diffusion-limiting membrane for limiting the diffusion of glucose is disposed instead of the polycarbonate membrane to increase the glucose concentration measuring limit without dilution of a glucose solution.
The description hereinbefore which has discussed mainly the case of measuring the concentration of glucose, may be also applied to the case of measuring the concentration of other organic macromolecule, protein or the like.
In the enzyme electrode unit above-mentioned, when a solution containing a target substance to be measured also contains interfering substances having a large particle diameter, the diffusion-limiting membrane not only restricts the diffusion of the target substance to be measured, but also prevents the interfering substances from penetrating therethrough. This assures an accurate measurement of a wide range of concentrations of a target substance to be measured.
Upon completion of one measurement as above-mentioned, a relatively great amount of interfering substances stick to the diffusion-limiting membrane. This inevitably reduces that portion of the diffusion-limiting membrane which achieves a predetermined diffusion limitation for the target substance to be measured. Therefore, the diffusion-limiting membrane as it is, cannot assure an accurate measurement on and after the second operation. Accordingly, it is a common practice that, after a predetermined number of measurements has been made, preferably after every measurement has been made, the diffusion-limiting membrane is exchanged with a new one to achieve measurement without any influence of the interfering substances. If there are neither variations in the characteristics of the replaced diffusion-limiting membranes themselves, nor variations in diffusion-limiting membrane mounting condition, an accurate measurement can be assured with the influence of the interfering substances eliminated after the replacement of diffusion-limiting membrane.
However, it is not assured at all that such variations are absent. Generally, there exist not only considerable variations in the characteristics of diffusion-limiting membranes themselves, but also considerable variations in membrane mounting condition. Accordingly, even though the influence of the interfering substances can be eliminated, such variations may produce considerable variations in measured results.
Further, the membrane mounted on the base electrodes is extremely thin, requiring extreme care to be used when handling the membrane.
It may be proposed that the diffusion-limiting membrane is mounted on a cap or the like in consideration of its removal, and the diffusion-limiting membrane is adapted to be automatically stuck to the enzyme-immobilized membrane when such cap is secured to the electrode unit body, threadedly or in other manner.
If a diffusion-limiting membrane is mounted in such manner, the membrane has a limited portion for which physical adhesion is assured. Accordingly, when a plurality of measurements are made even without replacement of the diffusion-limiting membrane, the measured data may considerably vary.
More specifically, when no measurement is still made, the enzyme-immobilized membrane is held wet and the diffusion-limiting membrane is also held wet. However, since no excessive electrode conserving liquid is present, the adhesion of the enzyme-immobilized membrane to the diffusion-limiting membrane is assured fairly well throughout the surfaces. However, when measurement starts by dropping a target solution to be measured on the diffusion-limiting membrane or by dipping the enzyme electrode unit in such solution, both membranes become excessively wet due to the target solution to be measured. Accordingly, it is considered that the adhesion of both membranes at other portions thereof than those to which a physical pressing force is directly applied, may be destroyed under the influence of surface tension or the like. It is also considered that the adhesion of both membranes may be destroyed under the influence of target solution dropping conditions or conditions of dipping the enzyme electrode unit in the target solution. The extent to which the adhesion of both membranes is destroyed, varies in each measurement, resulting in variations in measured data as above-mentioned.
Further, the diffusion-limiting membrane itself is very thin. This requires extreme care to be used when replacing or handling the membrane.